Acetyl coenzyme A (acetyl-CoA) is a key intermediate in the synthesis of essential biological compounds, including polyketides, fatty acids, isoprenoids, phenolics, alkaloids, vitamins, and amino acids. Among the metabolites derived from acetyl-CoA are primary and secondary metabolites, including compounds of industrial utility. In yeast, acetyl-CoA is biosynthesized from pyruvate metabolism (FIG. 1). However, in this biosynthetic pathway, CO2 is lost via the reactions catalyzed by pyruvate carboxylase and/or pyruvate dehydrogenase. In an industrial fermentation setting, one benefit of providing an alternative to pyruvate metabolism and lower glycolysis is that less CO2 is produced in the decarboxylation of pyruvate, and thus more carbon can be captured in the end product, thereby increasing the maximum theoretical yield. A second benefit is that less NADH is produced, and therefore significantly less oxygen is needed to reoxidize it. This can be accomplished by expressing phosphoketolase (PK; EC 4.1.2.9) in conjunction with phosphoacetyltransferase (PTA; EC 2.3.1.8).
PK and PTA catalyze the reactions to convert fructose-6-phosphate (F6P) or xylulose-5-phosphate (X5P) to acetyl-CoA. As shown in FIG. 1, PK draws from the pentose phosphate intermediate xyulose 5-phosphate, or from the upper glycolysis intermediate D-fructose 6-phosphate (F6P). PK splits X5P into glyceraldehyde 3-phosphate (G3P) and acetyl phosphate, or F6P into erythrose 4-phosphate (E4P) and acetyl phosphate. PTA then converts the acetyl phosphate into acetyl-CoA. G3P can re-enter lower glycolysis, and E4P can re-enter the pentose phosphate pathway or glycolysis by cycling through the non-oxidative pentose phosphate pathway network of transaldolases and transketolases.
The applicants have previously described the improved efficiency of heterologous isoprenoid production that can be gained with the introduction of PK and PTA enzymes. See U.S. application Ser. No. 13/673,819 (now U.S. Pat. No. 8,415,136), filed on Nov. 9, 2012, the contents of which are hereby incorporated by reference in their entirety. In particular, when cytosolic acetyl-CoA is synthesized from glucose using only the chemical reactions which occur in the native yeast metabolic network, the maximum possible stoichiometric yield for conversion of glucose to the isoprenoid farnesene via the mevalonate pathway is 23.6 wt %. By including the reactions catalyzed by acetaldehyde dehydrogenase, acetylating (ADA; EC 1.2.1.10) and NADH-using HMG-CoA reductase into the metabolic network for mevalonate production, the maximum theoretical stoichiometric yield is improved to 25.2 wt %. With the further introduction of PK and PTA, the reaction network, at optimality, is able to reach 29.8 wt % mass yield or greater, a significant increase in maximum theoretical yield.
Sondregger et al. have also described the benefits of PK and PTA with respect to ethanol production in a xylose-utilizing yeast strain. See Sondregger et al., Applied and Environmental Microbiology 70(5):2892-2897 (2004), the contents of which are hereby incorporated by reference in their entirety. The heterologous phosphoketolase pathway (PK, PTA, and ADA) was introduced in S. cerevisiae to address low ethanol yields that result from overexpression of NAD(P)H-dependent xylose reductase and NAD+-dependent xylitol dehydrogenase from Pichia stipitis. The different cofactor preferences in the two oxidoreductase reactions caused an anaerobic redox balancing problem that manifested in the extensive accumulation of the reduced reaction intermediate xylitol, and thus, low ethanol yields. Redox metabolism was balanced by introducing the phosphoketolase pathway, which lead to the net reoxidation of one NADH per xylose converted to ethanol, and an improvement in ethanol yield by 25%. However, overexpression of PK also leads to an increase in acetate accumulation and a reduction in fermentation rate. Although some acetate accumulation could be reduced by combining the phosphoketolase pathway with a mutation of ALD6, which converts acetaldehyde to acetate, the flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. The authors suggested that higher activities of phosphotransacetylase and/or acetaldehyde dehydrogenase may be necessary to prevent phosphoketolase pathway-based acetate formation.
Thus, while the introduction of a heterologous PK pathway can lead to substantial improvements in the yields of acetyl-CoA derived compounds, further improvements in the implementation of this pathway appear to be required to achieve optimal carbon flux through PK and PTA. The compositions and methods provided herein address this need and provide related advantages as well.